Покращення характеристик методик високоефективної рідинної хроматографії для визначення малих кількостей речовин у змивах при валідації відмивання.

The solvation parameter model is used to elucidate the retention mechanism of neutral compounds on the pentafluorophenylpropylsiloxane-bonded silica stationary phase (Discovery HS F5) with methanol-water and acetonitrile-water mobile phases containing from 10 to 70% (v/v) organic solvent. The dominant factors that increase retention are solute size and electron lone pair interactions while polar interactions reduce retention. A comparison of the retention mechanism with an octadecylsiloxane-bonded silica stationary phase based on the same silica substrate and with a similar bonding density (Discovery HS C18) provides additional insights into selectivity differences for the two types of stationary phase. The methanol-water solvated pentafluorophenylpropylsiloxane-bonded silica stationary phase is more cohesive and/or has weaker dispersion interactions and is more dipolar/polarizable than the octadecylsiloxane-bonded silica stationary phase. Differences in hydrogen-bonding interactions contribute little to relative retention differences. For mobile phases containing more than 30% (v/v) acetonitrile selectivity differences for the pentafluorophenylpropylsiloxane-bonded and octadecylsiloxane-bonded silica stationary phases are no more than modest with differences in hydrogen-bond acidity of greater importance than observed for methanol-water. Below 30% (v/v) acetonitrile selectivity differences are more marked owing to incomplete wetting of the octadecylsiloxane-bonded silica stationary phase at low volume fractions of acetonitrile that are not apparent for the pentafluorophenylpropylsiloxane-bonded silica stationary phase. Steric repulsion affects a wider range of compounds on the octadecylsiloxane-bonded than pentafluorophenylpropylsiloxane-bonded silica stationary phase with methanol mobile phases resulting in additional selectivity differences than predicted by the solvation parameter model. Electrostatic interactions with weak bases were unimportant for methanol-water mobile phase compositions in contrast to acetonitrile-water where ion-exchange behavior is enhanced, especially for the pentafluorophenylpropylsiloxane-bonded silica stationary phase. The above results are compatible with a phenomenological interpretation of stationary phase conformations using the haystack, surface accessibility, and hydro-linked proton conduit models.

A simple, sensitive, precise and accurate HPTLC method has been developed for the simultaneous estimation of the ciprofloxacin and ornidazole in tablets. The stationary phase used was precoated silica gel 60F 254 plate. The mobile phase used was a mixture of n-butanol:ethanol:8M ammonia (5:0.5:1.5, v/v/ v). The densitometric scanning was carried out at 315 nm. This system was found to give compact spots for ciprofloxacin (R f value of 0.25 0.004) and ornidazole (R f value of 0.82 ± 0.007). The method was validated in terms of linearity, accuracy, precision, limit of detection, limit of quantification and specificity in accordance with the ICH guidelines. The calibration curve was found to be linear between 40 to 140 ng/spot for each ciprofloxacin and ornidazole with significantly high value of correlation coefficient (r 2 > 0.99). The limit of detection and quantitation were found to be 10.01 and 33.03 ng/spot, respectively for ciprofloxacin and the limit of detection and quantification were found to be 7.62 and 25.15 ng/spot, respectively for ornidazole. The results of analysis have been validated statistically and by recovery studies. The proposed HPTLC method can be successfully applied in the routine analysis of commercial pharmaceutical tablets.

The solvation parameter model is used to elucidate the retention mechanism on a perfluorohexylpropylsiloxane-bonded (Fluophase RP) and octadecylsiloxane-bonded (Betasil C18) stationary phases based on the same silica substrate with acetonitrile–water and methanol–water mobile phase compositions. Dewetting affects the retention properties of Fluophase RP at mobile phase compositions containing less than 20% (v/v) acetonitrile or 40% (v/v) methanol. It results in a loss of retention due to an unfavorable change in the phase ratio as well as changes in specific intermolecular interactions. Steric repulsion reduces retention of bulky solutes on fully solvated Betasil C18 with methanol–water (but not acetonitrile–water) mobile phase compositions but is not important for Fluophase RP. The retention of weak bases is affected by ion-exchange interactions on Fluophase RP with acetonitrile–water, and to a lesser extent, methanol-water mobile phases but these are weak at best for Betasil C18. The system constants of the solvation parameter model and retention factor scatter plots are used to compare selectivity differences for Fluophase RP, Betasil C18 and a perfluorophenylpropylsiloxane-bonded silica stationary phase Discovery HS F5 for conditions where incomplete solvation, steric repulsion and ion-exchange do not significantly contribute to the retention mechanism. Lower retention on Fluophase RP results from weaker dispersion and/or higher cohesion moderated to different extents by polar interactions since solvated Fluophase RP is a stronger hydrogen-bond acid and more dipolar/polarizable than Betasil C18. Retention factors for acetonitrile–water mobile phases are highly correlated for Fluophase RP and Betasil C18 except for compounds with a large excess molar refraction and weak hydrogen-bonding capability. Selectivity differences are more significant for methanol–water mobile phases. Retention factors on Fluophase RP are strongly correlated with those on Discovery HSF5 for acetonitrile–water mobile phases while methanol–water mobile phases retention on Fluophase RP is a poor predictor of the retention order on Discovery HS F5.

126-P

PurposeDual‐energy computed tomography (DECT) has been proposed for quantification of hepatic iron concentration (IC). However, the lower limit of quantification (LLOQ) has not been established, limiting the clinical adoption of this technology. In this study, we aim to (a) establish the LLOQ using phantoms and (b) investigate the effects of patient size, dose level, energy combination, and reconstruction method.MethodsThree phantom sizes and eight vials of ferric nitrate solution with IC ranging from 0 to 10 mg/ml were used. DECT scans were performed at 80/140 and 100/140Sn kVp, and using five different levels of CT dose index (CTDI). An image‐domain three‐material‐decomposition algorithm was used to calculate the IC. The LLOQ was determined based on the coefficient of variation from repeated measurements.ResultsThe measured IC correlated strongly with the true IC in the small and medium phantoms (R2 of linear regression > 0.99) and moderately in the large phantom (0.8 < R2<0.9). The LLOQ improved with increased CTDI. At 30 mGy, the LLOQ was found to be 0.50/1.73/6.25 mg/ml in the small/medium/large phantoms, respectively. 80/140Sn kVp resulted in superior LLOQ for all phantom sizes compared to 100/140Sn kVp, primarily due to the difference in their iron enhancement ratios (1.94 and 1.55, respectively). Iterative reconstruction was found to further improve the LLOQ (by ~ 11%), whereas reconstruction kernel smoothness had negligible effect. The LLOQ of iron was significantly higher than that of iodine due to its lack of a useful k‐edge and lower enhancement ratio.ConclusionIron quantification at clinically important levels was achieved in a small‐ and a medium‐sized phantom using DECT, but proved challenging in a large phantom. Wide spectral separation and accurate calibration were found to be critical to the success of the technology.

Diosmin is a flavonoid often administered in the treatment of chronic venous insufficiency, hemorrhoids, and related affections. Diosmin is rapidly hydrolized in the intestine to its aglicone, diosmetin, which is further metabolized to conjugates. In this study, the development and validations of three new methods for the determination of diosmetin, free and after enzymatic deconjugation, and of its potential glucuronide metabolites, diosmetin-3-O-glucuronide, diosmetin-7-O-glucuronide, and diosmetin-3,7-O-glucuronide from human plasma and urine are presented. First, the quantification of diosmetin, free and after deconjugation, was carried out by high-performance liquid chromatography coupled with tandem mass spectrometry, on an Ascentis RP-Amide column (150 × 2.1 mm, 5 μm), in reversed-phase conditions, after enzymatic digestion. Then glucuronide metabolites from plasma were separated by micro-liquid chromatography coupled with tandem mass spectrometry on a HALO C18 (50 × 0.3 mm, 2.7 μm, 90 Å) column, after solid-phase extraction. Finally, glucuronides from urine were measured using a Discovery HSF5 (100 × 2.1 mm, 5 μm) column, after simple dilution with mobile phase. The methods were validated by assessing linearity, accuracy, precision, low limit of quantification, selectivity, extraction recovery, stability, and matrix effects; results in agreement with regulatory (Food and Drug Administration and European Medicines Agency) guidelines acceptance criteria were obtained in all cases. The methods were applied to a pharmacokinetic study with diosmin (450 mg orally administered tablets). The mean Cmax of diosmetin in plasma was 6,049.3 ± 5,548.6 pg/mL. A very good correlation between measured diosmetin and glucuronide metabolites concentrations was obtained. Diosmetin-3-O-glucuronide was identified as a major circulating metabolite of diosmetin in plasma and in urine, and this finding was confirmed by supplementary experiments with differential ion-mobility mass spectrometry.

Ghrelin is a major appetite-stimulating hormone. It circulates as acylated ghrelin (AG) and unacylated ghrelin (UAG), which could have different metabolic actions in obesity. Our objective was to study the analytical performance of two new canine AG and UAG ELISAs using blood samples from healthy, normal-weight dogs. Additionally, the effect of a protease inhibitor (PI) on short-term sample storage was analyzed. The intra- and inter-assay precision for low, intermediate, and high AG and UAG concentrations, accuracy, limits of quantification (LQ), and detection limit (DL) of a blank sample were determined in ten healthy dogs. To study the effects of a PI on ghrelin concentrations, andAG and UAG concentrations were compared in five canine plasma samples stored for 1month with (PI+), without (PI-), and with PI added at sample thawing (PI+th). The intra- and inter-assay coefficients of variation were 1.8%-5.7% and 2.9%-6.4% for the AG assay, and 0.8%-7.5% and 2.8%-13.4% for the UAG assay, respectively. Accuracy analyses showed nonsignificant deviation from linearity for the AG (R2 =.99; Runs test: P=.37) and UAG (R2 =.99; Runs test: P=.42) assays. For the AG assay, the upper LQ was >1261pg/mL, the lower LQ was 6.2pg/mL, and the DL was 0.3pg/mL. For the UAG assay, the upper LQ was >1785pg/mL, the lower LQ was 16.3pg/mL, and the DL was 1.8pg/mL. No differences in the AG (P=.54) and UAG (P=.95) concentrations were detected in the plasma samples subjected to PI+, PI-, and PI+th. The AG and UAG ELISA assays had acceptable precision, accuracy, lower LQ, and DLs, but upper LQ could not be established. An influence of the PI on short-term storage was not detectable. Long-term storage±PI was not evaluated and should be investigated further.

The limit of detection (LOD) and the limit of quantification (LOQ) are common parameters to assess the sensitivity of analytical methods. In this study, the LOD and LOQ of previously reported terbium sensitized analysis methods were calculated by different methods, and the results were compared with sensitivity parameters [lower limit of quantification (LLOQ)] of U.S. Food and Drug Administration guidelines. The details of the calibration curve and standard deviation of blank samples of three different terbium-sensitized luminescence methods for the quantification of mycophenolic acid, enrofloxacin, and silibinin were used for the calculation of LOD and LOQ. A comparison of LOD and LOQ values calculated by various methods and LLOQ shows a considerable difference. The significant difference of the calculated LOD and LOQ with various methods and LLOQ should be considered in the sensitivity evaluation of spectroscopic methods.

Volatile fatty acids (VFA) are intermediate products during the anaerobic digestion process of complex waste. Accurate quantification of VFA is a useful measurement of the process. This study evaluated two extraction methods for gas chromatography determination of the following VFA: acetic acid, propionic acid, butyric acid, and valeric acid to find a simple and efficient preparation approaching on Green Analytical Chemistry methods. Tofu wastewater was used as the sample. The extraction was performed using diethyl ether and carried out either by vortex mixing or shaken by hand. The measurement was performed using gas chromatography with HP-INNOWax column and flame ionization detector. The linearity, precision, accuracy, limit of detection and limit of quantification were evaluated. VFA determination was linear at the concentration range of 5-500 μg/mL for all VFA. The precision of 2.59-12.63 % was obtained for both methods. The recovery of the fortified sample (10 µg-VFA/mL) was 75.33-98.31 % for vortex extraction and 99.81 to 103.80 % for shaken by hand extraction. Limits of detection and quantification of vortex extraction were 0.38-2.78 µg/mL and 1.06-3.03 µg/mL, respectively. Limits of detection and quantification using hand shaken extraction were 0.09-1.13 µg/mL and 0.55-1.61 µg/mL, respectively. Based on the high recovery and low limits of detection and quantification, shaken by hand extraction method can be used for rapid and accurate quantification of VFA.

More sensitive hepatitis C virus RNA detection: What for?

Citrus junos seeds (CS) have been traditionally used for the treatment of cancer and neuralgia. They are also used to manufacture edible oil and cosmetic perfume. A large amount of CS shells without oil (CSS) are discarded after the oil in CS is used as foods or herbal remedy. To efficiently utilize CSS as a by-products, it needs to be studied through chemical analysis. Therefore, we developed an ultra-performance liquid chromatography (UPLC)-diode array detection (DAD) method for simultaneous determination and quantitative analysis of five components (two flavonoids and threes limonoids) in CSS. A Waters Acquity UPLC HSS T3 column C18 (2.1 × 100 mm, 1.8 μm) was used for this separation. It was maintained at 40℃. The mobile phase used for the analysis was distilled water and acetonitrile with gradient elution. To identify the quantity of the five components, a mass spectrometer (MS) with an electrospray ionization (ESI) source was used. The regression equation showed great linearity, with correlation coefficient ≥ 0.9912. Limits of detection (LOD) and limits of quantification (LOQ) of the five compounds were 0.09 - 0.13 and 0.26 - 0.38 μg/mL, respectively. Recoveries of extraction ranged from 97.45% to 101.91%. Relative standard deviation (RSD) values of intra- and inter-day precision were 0.06 - 1.15% and 0.19 - 0.25%, respectively. This UPLC-DAD method can be validated to simultaneously analyze quantities of marker flavonoids and limonoids in CSS.

Development and comprehensive greenness assessment for MEKC and HPTLC methods for simultaneous estimation of sertaconazole with two co-formulated preservatives in pharmaceutical dosage forms

Improved separation of furocoumarins of essential oils by supercritical fluid chromatography

An analytical method using liquid chromatography tandem mass spectrometry equipped with electrospray ionization (ESI) was demonstrated for the determination of medicinal ingredients, such as fenfluramine (FF), Nnitrosofenfluramine (NFF), sibutramine (SIB), sildenafil (SDF), vardenafil (VDF), tadalafil (TDF) and xanthoanthrafil (XAF), in so-called health-promoting food. These analytes were clearly separated with acetonitrile-water (40 : 60) containing 4 mM formic acid and 8 mM ammonium formate used in the mobile phase on a pentafluorophenyl (PFP) column under isocratic conditions. The retention times of FF, SIB, SDF and VDF on the PFP column were longer than those on the C18 column under the same mobile phase conditions. Within wide ranges, all peaks were proportional and the coefficient of determination (r 2 ) showed more than 0.9950 in a linear regression analysis. The limit of quantification (LOQ) of the developed method was 0.8–42.2µg/l (S/N = 10). The recoveries of analytes admixed with commercially available health-promoting food ranged from 80.2 to 113.3% and were acceptable for quantitative analysis. Analytes of more than 2µg in a health-promoting food sample (0.5 g) were able to be identified by the European Communities (EC) criteria.

A specific, precise, and accurate LC-UV method was developed and validated to assay raloxifene hydrochloride in rat plasma. Raloxifene was analyzed after liquid-liquid extraction and quantified by reversed phase liquid chromatography (C18 column) using acetonitrile and ammonium acetate buffer 0.05 M (pH 4.0) as mobile phase at a flow rate of 1 mL.min-1 and UV detection at 287 nm. Retention times of raloxifene and internal standard (dexamethasone) were approximately 11 min and 14 min, respectively. Linearity was checked for a concentration range between 25 ng.mL-1 and 1000 ng.mL-1. Intra- and inter-day precision had relative standard deviation lower than 10% and 15%, respectively. Recovery from plasma was higher than 90%. Accuracy values were 98.21%, 99.70%, and 102.70% for lower, medium, and upper limits of quantification, respectively. Limit of quantification was 25 ng.mL-1. Drug stability was analyzed at room temperature using plasma kept in a freezer at -80 °C for 45 days after processing for 6 h and three freeze-thaw cycles. The advantages of the method developed include stability under different conditions and low limit of quantification. Its applicability was confirmed by the analysis of raloxifene levels in plasma samples in a designed pharmacokinetic study in rats after intravenous administration (5 mg.kg-1).